1. Field of the Invention
The subject of the invention is the strain composition of the Lactobacillus genus and the application of the strain composition of the Lactobacillus genus in the production of the pharmaceutical preparation.
2. Brief Description of the Background of the Invention Including Prior Art
The composition of the vaginal microflora is diverse and depends on the stage of life a woman is in and is strictly dependent on hormonal activity of the body. Basic knowledge of the vaginal ecosystem was put forward by Cruickshank and Sherman, who described the vaginal microflora of the fetus in the uterus, of infants, girls before puberty, and women in the reproductive phase and after menopause. In the uterus the fetal vagina is sterile, however, 2-3 weeks after birth the quantitative composition of microflora is almost the same as the quantitative composition of microflora of an adult woman's vagina, which is the consequence of estrogen stimulation by the mother. Before the occurrence of the first menstruation, vaginal environment is dominated by bacteria such as S. epidermidis, Bacteroides spp., Peptococcus spp., Porphyromonas spp., G. vaginalis. During the reproductive period the vaginal environment is mostly dominated by bacterial population of the Lactobacillus genus. In the past it was believed that this genus is represented by one strain only, that is Lactobacillus acidophilus. During the last decade the development of modern molecular methods allowed to sort out the classification of most of the bacteria, which proved that the Lactobacillus acidophilus species is not homogenous and is a part of a complex of a few other species such as L. crispatus, L. amylovorus, L. gallinarum, L. acidophilus sensu stricto, L. gasseri, L. johnsoni. Using modern molecular methods, especially the DNA sequencing technique, as well as modified culturing procedure it was proven that the most common strains found in the natural vaginal environment of healthy women are some various species, which are a part of the acidophilus complex such as L. johnsoni, L. gasseri, L. acidophilis, L. crispatus, and above all L. plantarum and L. fermentum. 
The mechanisms by which the bacteria from the genus Lactobacillus effect the vaginal environment are not yet fully understood. However, it is widely known that they:                synthesize antimicrobial substances, such as lactic, acetic, pyroglutamic acids, in order to lower the pH of the vaginal environment to obtain the values of 3.7-4.6,        synthesize short peptides and/or proteins with a very complex structure, resembling bacteriocins, which adhere to the receptors at the surface of susceptible bacteria leading to their lyses and eventually their death,        compete with other microorganisms, especially pathogenic, for nutritive substances and for the epithelial receptors of the vagina,        modify toxins and toxins' receptors,        produce hydrogen peroxide—some Lactobacillus strains, which produce H2O2, dominate the microbe population of healthy vagina,        exert different immuno-modulating effects on the host's immunological system.        
Due to these many different properties, the bacteria of the Lactobacillus genus are able to maintain a certain biological state of equilibrium of the vagina, preventing the numerous population growth of pathogenic microorganisms residing in the woman's genital tract, which also decreases the risk of occurrence of bacterial vaginosis.
Due to the fact that most of infections of the genitourinary tract in women are caused by the imbalance of the vaginal microflora, the ways of prevention as well as treatment are directed towards restoration of correct microbial balance.
In 1992, in the United States an experiment was carried out (Hilton E, Isenberg H D, Alperstein P, France K, Borenstein M T. Ingesting of yogurt containing Lactobacillus acidophilus as prophylaxis for candidal vaginitis. Ann Itern. Med 1992; 116: 353-357), which involved a consumption of yogurt containing Lactobacillus acidophilus on daily basis for 6 months by 35 women with recurring candidiasis of vulva and vagina in order to prevent the recurring illness. The endpoint of the experiment was colonization of vagina and anus by Candida and Lactobacillus as well as candidiasis. Thirteen patients finished the experiment, however, the analysis was done for 33 of them. During the 6-month period a relevant decrease in the number of infections caused by Candida was found in persons who were colonized (anus and vagina) by Candida. An experiment was done with the use of a regular yogurt containing L. acidophilus, which produced a moderate quantity of H2O2. The strains of L. acidophilus isolated from the women were not identified. However, a close relationship between the presence of L. acidophilus in the anus and vagina was observed: if L. acidophilus was absent in the anus, the probability of its presence in the vagina was 15.5%, on the other hand, if present in the anus, the probability of its presence in the vagina was 54.8% (P<0.001). The authors concluded that the intestinal strain of L. acidophilus was colonizing the vagina, however, due to the lack of molecular evidence, it was impossible to prove.
A similar experiment was done in 1996 in Israel (Shalev E, Battino S, Weiner E, Colodner R, Keness Y. Ingestion of yogurt containing Lactobacillus acidophilus compared with pasteurized yogurt as prophylaxis for recurrent candidal vaginitis and bacterial vaginosis. Arch Fam Med 1996; 5: 593-596) based on the same protocol and with the use of yogurt containing L. acidophilus producing H2O2. The data obtained in this experiment did not show a prophylactic activity on the recurrent vaginal blastomycosis (the same effect was shown by the yogurt containing dead bacteria), however, it showed an effect on bacterial vaginosis, at the same time increasing the number of women colonized by L. acidophilus. Also, a coexisting colonization of the anus and vagina by Candida was observed.
In 2001, in Finland (Kontiokari T, Sundqvist K, Nuutinen M, Pokka T, Koskela M, Uhari M. Randomized trial of cranberry-lingonberry juice and Lactobacillus GG drink for the prevention of urinary tract infections in women. Brit Med J 2001; 322: 1571-1573) an open randomized experiment on the possibility of preventing recurring urinary tract infections in women was carried out with the use of cranberry and lingonberry juice or Lactobacillus GG drink. The experiment lasted for 12 months and involved the observation of recurring infections in 150 women divided into 3 groups, who were earlier diagnosed with an urinary tract infection (>105 bacteria/ml of urine). The final parameter of the experiment was the observation of a recurring infection. As compared to the cranberry/lingonberry juice, a positive effect of Lactobacillus GG was not observed. The cause of this may be that Lactobacillus GG, being a typical intestinal strain, does not colonize the urethra opening/vagina.
In 2003 (Reid G, Charbonneau D, Erb J, Kochanowski B, Beuerman D, Poehner R, Bruce A W. Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trail in 64 healthy women. FEMS Immunol Med Microbiol 2003; 35: 131-134) a randomized clinical experiment with the use of placebo was carried out in order to check the effect of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 orally administered for 60 days on vaginal flora of 64 healthy women. Microscopic analysis showed that 37% of the examined women returned to their normal flora compared to the 13% of women from the control group. The culture showed a relevant increase in the number of Lactobacillus, and a decrease in the number of yeasts and intestinal rods. The probiotic was considered safe upon daily administration.
In 2004, the same authors carried out another experiment where the same strains (Reid G, Burton J, Hammond J A, Bruce A W. Nucleic acid-based diagnosis of bacterial vaginosis and improved management using probiotic lactobacilli. J Med Food 2004; 7: 223-228) were administered to 59 women with non-symptomatic bacterial vaginosis diagnosed with the help of DGGE. After 2 months of probiotic administration, the restoration of the microbial flora of the vagina, dominated by Lactobacillus, was shown.
Migration (cross-colonization) of bacteria from the anus to vagina also involves pathogens such as: Streptococcus agalactiae, uropathogenic strains of Escherichia coli or Staphylococcus aureus. In the recently published paper (Warner J E, Onderdonk A B. Diversity of toxic shock syndrome toxin I-positive Staphylococcus aureus isolates. Appl Environ Microbiol 2004; 70: 6931-6935) it was shown, with the use of the PFGE method, that the same strains of S. aureus are present in both places in 59% of examined women.
It should be noted that there is a lack of proof in literature for migration or cross-colonization of the anus and vagina by the same strains of Lactobacillus. This may be due to the fact that in the past the adequate molecular typing methods were not known. Additionally, the administration of the Lactobacillus strains with dairy products is not possible in all the patients due to dietary reasons. The described Lactobacillus strains, administered orally, showed inadequate survival in the digestive tract and required protection of special capsules.
It is known from the Polish patent's description no. 189577, the use of pairing between lactic acid bacteria of the strain Lactobacillus brevis and Lactobacillus salivarius subspecies salicinius for production of pharmaceutical composition used for vaginal administration in order to treat vaginosis and vaginal infections. The addressed patent's description also reveals the pharmaceutical composition containing the paired lactic acid bacteria of the strains Lactobacillus brevis and Lactobacillus salivarius subspecies salicinius. Based on the obtained data (Strus M., The basics in using probiotics administered into the vagina during the infections of genitourinary tract. Infections, 2005, 4, 40-43) it is concluded that vaginal microflora of healthy women is mainly composed of a few strains of Lactobacillus. It was shown that the strains isolated most often were the strains of the L. acidophilus complex, as well as the strains: L. fermentum and L. plantarum. The strains used, based on the patent's description no. 189577, were not characteristic for the Polish women population. Additionally, the form of administration (vaginal) is uncomfortable for daily use.
There are new strains known from the Polish patent's description no. P-373518: Lactobacillus fermentum 57A deposited under number-B/00007, Lactobacillus plantarum 57B deposited under number-B/00008, Lactobacillus gasseri 57C deposited under number-B/00009, Lactobacillus plantarum 78B deposited under number-B/00010.
The subject of the invention is also the composition of new strains of the genus Lactobacillus, which includes strains Lactobacillus fermentum 57A, Lactobacillus plantarum 57B, Lactobacillus gasseri 57C, with relative numbers in the proportion of 0.5:0.5:1.
The above-said strains were obtained from the posterior vaginal vault of a healthy woman who had a normal vaginal flora. The performed tests showed that the strains Lactobacillus plantarum 57B, Lactobacillus fermentum 57A, Lactobacillus gasseri 57C comply with all the requirements laid down to probiotic products. Based on the performed tests it may be concluded that the new strains are more favorable in comparison to the other tested strains. In addition, each one of the selected and deposited strains possesses a strong leading characteristic. The strain Lactobacillus plantarum 57B is characteristic for strong antagonistic features especially for pathogens such as: Gardnerella vaginalis and Escherichia coli, Lactobacillus fermentum 57A adheres very well to the epithelium of the host, and Lactobacillus gasseri 57C produces large amounts of hydrogen peroxide.